The long term goal of research in my laboratory has been to develop small animal models where aspects of HIV-1 pathogenesis can be studied. Recently we have constructed strains of transgenic mice that express the HIV-1 nef gene in T cells. Development of CD4+ T cells is defective in these CD3 Nef1 animals. Transgenic T cells show altered responses to T cell receptor stimulation and CD4 antigen expression on the cell surface is abnormally low. Evidence from these animals strongly suggests that nef blocks the normal CD4 function in CD4+ T cells. These effects of nef are relevant to human AIDS because we have shown that HIV-1 nef alleles isolated directly from HIV-1 infected patients down-regulate CD4 expression in human T cells. Therefore it is very likely that nef expression has similar consequences for both human and murine T cells and that transgenic mice will provide valid animal models to study nef gene function in primary T cells. The main focus of this proposal is to use the transgenic mouse system (i) to define the effect of HIV-l Nef on the normal function(s) of primary murine T cells and (ii) to begin dissecting the cellular signalling mechanisms involved. In particular we will test the hypothesis that Nef can counter-act the negative effects of CD4 on T cell activation and CD4- mediated induction of apoptosis. The first set of experiments will address the relation between the enhanced CD3/T cell receptor signalling in thymic T cells from CD3 Nef1 animals and the increased presence of these cells in vivo. We will assess whether Nef promotes survival of these cells. The second set of genetic experiments will assess whether Nef perturbs T cells via CD4 and/or via other mechanisms. A mutant CD4 protein that does not interact with nef will be constructed and expressed in transgenic mice. Genetic experiments in transgenic mice will assess whether CD4-n(-) rescues normal T cell development and activation in the presence of Nef in CD3 Nef1 mice. Finally, the third set of experiments will assess whether Nef can counteract the cytotoxic effect of gpl60/120-CD4 complexes in established T cell lines in vitro, and in vivo in transgenic mice. These effects could provide an explanation far (i) previously observed strict requirement for nef function for high viral load and pathogenesis in the SlV/rhesus monkey model of human AIDS and (ii) lack -of a similar requirement for viral replication in vitro. Understanding the mechanism of nef action may lead to novel preventive and/or therapeutic strategies for HIV infection.